When the uracil analog 5-fluorouracil (FU) is added to a rapidly growing culture of Escherichia coli B or Saccharomyces cerevisae, extensive replacement of uracil or modified nucleosides derived from uridine in tRNA occurs. In E. coli, the minor nucleosides replaced include pseudouridine, ribothymidine, 5,6-dihydrouridine, 4-thiouridine, 5-methylamino-methyl-2-thiouridine and uridine-5-oxyacetic acid. We plan to compare the properties of normal and FU-containing tRNAs, having up to about 90% of the uracils replaced by FU in an effort to gain possible insights into the role of these minor components. This type of study can provide valuable information in regard to what minor bases or areas of tRNA molecules can be altered without changing structure and function. The biological functions to be compared include aminoacylation, codon recognition, efficiency of codon response, the ability to support peptide synthesis in vitro, and in vitro methylation. RNAs extracted from E. coli treated with 2-thiouracil are being examined for structural and functional anomalies.